The overall goal of this research proposal is to gain a fundamental understanding of insulin receptor metabolism (synthesis and degradation), and elucidate the mechanism(s) mediating insulin-induced cell-surface receptor regulation in physiological and hyperinsulinemic states. To achieve this goal, we plan to couple our recently developed technique for maintaining isolated adipocytes in primary culture with established methods for biosynthetically and externally labeling insulin receptors. In addition, we intend to use our technique for rapidly and accurately determining the cellular distribution of receptors. This methodology will subserve four specific aims: (1) To delineate the insulin receptor biosynthetic pathway. This will include studies on the structure and function of receptors during processing and maturation, the time-course of receptor transit, and post-translational modification of receptors. (2) To explore the receptor degradative pathway. Specifically, we plan to establish receptor turnover rates for both cell-surface and newly synthesized receptors, and examine how and where cell-surface receptors are degraded. (3) To elucidate the mechanism(s) by which insulin regulates the net number of cell-surface receptors. This will be accomplished by determining whether insulin-induced changes in surface receptors (up-and down-regulation) are mediated by changes in the rate of receptor synthesis, receptor degradation, or by a redistribution of receptors (cell-surface vs. intracellular). Other studies will explore the lag-time preceding insulin-induced receptor loss, with special emphasis on the characteristics and mechanisms by which insulin "triggers" cellular events so that receptor regulation can be manifested in the absence of ligand. Additional experiments will attempt to separate genetic control from environmental regulation of insulin receptors. And lastly, (4) to confirm that insulin regulates the rate at which insulin receptors are endocytosed, and explore the mechanism involved in this process. This will include studies on the specificity of this affect, and the site of regulation (receptor vs. post-receptor regulation). Hopefully, these combined studies will provide new insights into the mechanism of insulin action and advance our understanding of certain pathophysiological, insulin resistant states characterized by hyperinsulinemia and receptor down-regulation, such as in obesity and Type II diabetes mellitus.